** This formula is applicable to [http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CDEQFjAA&url=http%3A%2F%2Fwww.neb.com%2Fnebecomm%2Fproducts%2Fproductm0530.asp&ei=8NV0UL6-BaP1igLzsoGABQ&usg=AFQjCNEAXwNrh7KmjNNLXmdlaS1n74xitw&sig2=hnZPEtJmOMyRtqLdiSe_IQ Phusion DNApolymerase], the DNA polymerase used to form the DNA you will assemble.

** This formula is applicable to [http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CDEQFjAA&url=http%3A%2F%2Fwww.neb.com%2Fnebecomm%2Fproducts%2Fproductm0530.asp&ei=8NV0UL6-BaP1igLzsoGABQ&usg=AFQjCNEAXwNrh7KmjNNLXmdlaS1n74xitw&sig2=hnZPEtJmOMyRtqLdiSe_IQ Phusion DNApolymerase], the DNA polymerase used to form the DNA you will assemble.

* Use cheap primers

* Use cheap primers

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** If ordering with [http://www.idtdna.com/site IDT], primers should be 60 bp if you are encoding homology. The price per base pair jumps when you add the 61st base pair: we pay ~$9 for a 60 bp primer but ~ $34 for a 61 bp primer. Using less than 60 bp lessens the base pairs of homology between adjacent DNA pieces in the assembly. There are cases when you use standard size (18-22 bp) primers as is discussed in this page. *** DISCUSS ***

+

** If ordering with [http://www.idtdna.com/site IDT], primers should be 60 bp if you are encoding homology. The price per base pair jumps when you add the 61st base pair: we pay ~$9 for a 60 bp primer but ~ $34 for a 61 bp primer. Using less than 60 bp reduces the length of the homolgy between adjacent DNA pieces in the assembly. Note: there are cases when you use standard size (18-22 bp) primers as is discussed in this page. *** DISCUSS ***

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* Check primers for cross dimers with Finnzyme's [http://www.google.com/url?q=http%3A%2F%2Fwww.finnzymes.fi%2Fjava_applets%2Fmultiple_primer_analyzer.html&sa=D&sntz=1&usg=AFQjCNGBTnwPK8yo48XqV5kq14dVTMt4sg multiple primer analyzer]. If the annealing temperature of the primer dimer(s) is low, this will probably not be a problem during PCR.

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* Make sure the reverse primer is reverse complemented!

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=== Double Check your Design ===

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* Blast your primers and templates with [http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Nucleotides&PROGRAM=blastn&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&BLAST_SPEC=blast2seq&QUERY=&SUBJECTS= blastn] and make sure they only anneal where you expect.

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* Blast the APE files for the expected PCR products against each other to make sure they have the correct amount of overlap. Make sure there is not extensive homology in other places.

=== Generate PCR fragments ===

=== Generate PCR fragments ===

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*I run each PCR at a few annealing temps and DMSO concentrations. See my sample spreadsheet.

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*[http://www.neb.com/nebecomm/products/productr0176.asp Dpn1 can be added after the PCR is complete to degrade the template DNA. This will reduce the number of background colonies when you transform.

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* Run a few uL of each PCR product on a gel to identify rxn conditions that yield a lot of product.

Procedure

Make a plasmid map of your design

This is key. You will want it for primer design, checking your primers, assessing sequencing reactions, etc. I use APE, open-source software. See my APE use page

Design primers

The primers should confer 20-100 bp of homology between to adjacent overlapping segments. 40 - 100 bp is ideal; substantially shorter or longer will give you lower yields.

The annealing portion of the primer should have Tm between 62oC and 65oC as calculated by this Finnzymes website

This formula is applicable to Phusion DNApolymerase, the DNA polymerase used to form the DNA you will assemble.

Use cheap primers

If ordering with IDT, primers should be 60 bp if you are encoding homology. The price per base pair jumps when you add the 61st base pair: we pay ~$9 for a 60 bp primer but ~ $34 for a 61 bp primer. Using less than 60 bp reduces the length of the homolgy between adjacent DNA pieces in the assembly. Note: there are cases when you use standard size (18-22 bp) primers as is discussed in this page. *** DISCUSS ***

Check primers for cross dimers with Finnzyme's multiple primer analyzer. If the annealing temperature of the primer dimer(s) is low, this will probably not be a problem during PCR.

Make sure the reverse primer is reverse complemented!

Double Check your Design

Blast your primers and templates with blastn and make sure they only anneal where you expect.

Blast the APE files for the expected PCR products against each other to make sure they have the correct amount of overlap. Make sure there is not extensive homology in other places.

Generate PCR fragments

I run each PCR at a few annealing temps and DMSO concentrations. See my sample spreadsheet.